Systems, methods, and devices for sterilizing antiseptic solutions

ABSTRACT

A method for sterilizing an antiseptic solution includes providing a container containing the antiseptic solution, the antiseptic solution having an initial purity, selecting a sterilization temperature from about 85° C. to about 135° C. and an sterilization time from about 1 minute to about 19 hours, heating the antiseptic solution to the selected sterilization temperature, maintaining the temperature for the selected sterilization time, and terminating the heating of the antiseptic solution when the sterilization time expires. After terminating the heating, the antiseptic solution has a post-sterilization purity. The sterilization temperature and the sterilization time are selected such that after terminating the heating, the antiseptic solution is sterile and has a post-sterilization purity of at least about 92% and the percentage point change in purity from the initial purity to the post-sterilization purity is at most about 5%.

FIELD OF THE INVENTION

Aspects of the present invention relate to the field of sterilization,and in particular, to sterilization of topical antiseptic solutions.

BACKGROUND OF THE INVENTION

In the United States there are currently no regulations regarding thesterilization requirements of topical antiseptic solutions. Therefore,antiseptic solutions currently sold in the United States generally donot undergo a sterilization process. In other jurisdictions, however,such as European Union (EU) countries, some degree of sterilization isrequired. A known antiseptic solution containing 2% w/v chlorhexidinegluconate in 70% v/v isopropanol in water, manufactured by CareFusionCorp., is sterilized for EU countries using a known sterilizationmethod.

The known method of sterilization involves heat treating glass ampoulescontaining the chlorhexidine gluconate solution in a convection oven at76-80° C. for 24-31 hours. It is currently believed that relatively lowtemperature and relatively long processing time is necessary tosufficiently sterilize the solution without overly degrading theantimicrobial molecules, thereby avoiding reducing the concentration andpurity of the chlorhexidine gluconate contained therein as anantiseptic. Degrading the antimicrobial molecules creates undesiredimpurities as well as lowers the overall concentration of the activedrug moiety. Regulations in the United States and EU countries limit theamount of impurities that may be present in the antiseptic solution.Furthermore, a convection oven, which utilizes air to conduct heat, isan inefficient process because the initial heating of the solution fromroom temperature (also referred herein as “ramp up” time) and eventualcooling down of the solution back to room temperature (also referredherein as “cool down” time) is relatively long. For example the ramp uptime for the solution to reach the sterilization temperature can be 2-6hours while the cool down period can be 1-2 hours. Accordingly, the timethat the chlorhexidine gluconate solution is exposed to the sterilizingtemperature (i.e., the time at 76-80° C.) in the known method can be 22to 24 hours, while the total processing time (i.e., including ramp up,sterilization and cool down time) may be around 25 to 32 hours.

It is the industry belief that high temperature sterilization is notsuitable due to the expected degradation. See, for example, Kelly M.Pyrek, “Sterility of Antiseptic Products: FDA Investigates, Deliberateson Potential Recommendations,” Infection Control Today (July 2013):24-26 and Block, Seymour S. Disinfection, Sterilization, andPreservation. Philadelphia: Lippincott Williams & Wilkens, 322-323.2001.

Thus, there is an unmet need in the art for a method of sterilizingantiseptic solutions that has a shorter, more efficient processing time,and provides a sterile solution while maintaining sufficient purity ofthe antiseptic solution to comply with regulatory requirements.

SUMMARY OF THE INVENTION

Aspects of the present invention overcome the above identified problems,as well as others, by providing systems, methods, and devices forefficiently sterilizing antimicrobial solutions while maintainingantimicrobial efficacy as an antiseptic and purity of the active drugmoiety to comply with regulatory requirements.

In one example aspect the method for sterilizing an antiseptic solutioncomprises providing a container containing the antiseptic solution, theantiseptic solution having an initial purity; selecting a sterilizationtemperature from about 85° C. to about 135° C. and a sterilization timefrom about 1 minute to about 19 hours; heating the antiseptic solutionto the selected sterilization temperature; maintaining the antisepticsolution at the selected sterilization temperature for the selectedsterilization time; and terminating the heating of the antisepticsolution when the selected sterilization time expires. After terminatingthe heating, the antiseptic solution has a post-sterilization purity.The sterilization temperature and the sterilization time are selectedsuch that after terminating the heating, the antiseptic solution issterile and has a post-sterilization purity of at least about 92% andthe percentage point change in purity from the initial purity to thepost-sterilization purity is at most about 5%.

In another aspect, the sterilization temperature and sterilization timemay be selected such that the selected sterilization time and selectedsterilization temperature satisfy the following relationship:

-   -   a) 85° C.≤y<125° C.,        -   y≥−6.14·ln x+123.2 for 1≤x≤552, and        -   y≤−10.38·ln x+156.9 for 21.5≤x≤1123; or    -   b) 125° C.≤y≤135° C.,        -   x≥1 and        -   y≤−10.38·ln x+156.9 for 9.1≤x≤21.5,            wherein y is the sterilization temperature and x is the            sterilization time in minutes.

In another example aspect, the sterilization temperature and thesterilization time are selected such that after terminating the heating,the antiseptic solution has a post-sterilization purity of at leastabout 94% and the percentage point change in purity from the initialpurity to the post-sterilization purity is at most about 4%.

In another example aspect, the antiseptic solution comprises about 70%v/v isopropanol in water and about 2.0% w/v chlorhexidine gluconate.

In an another example aspect, the sterilization temperature is about 95°C. and the sterilization time is from about 90 minutes to about 6.5hours. In another aspect, the sterilization temperature is about 110° C.and the sterilization time is from about 6 minutes to about 90 minutes.In another aspect the sterilization temperature is about 120° C. and thesterilization time is from about 2 minutes to about 35 minutes.

In another example aspect, the selected sterilization temperature andthe selected sterilization time are chosen such that after terminatingthe heating, the antiseptic solution has a post-sterilization purity ofat least about 96% and the percentage point change in purity from theinitial purity to the post-sterilization purity is at most about 3%.

In another example aspect, the selected sterilization temperature andthe selected sterilization time are chosen such that after terminatingthe heating, the antiseptic solution has a post-sterilization purity ofat least about 98% and the percentage point change in purity from theinitial purity to the post-sterilization purity is at most about 2%.

Additional advantages and novel features relating to aspects of thepresent invention will be set forth in part in the description thatfollows, and in part will become more apparent to those skilled in theart upon examination of the following or upon learning by practicethereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of sterilization temperature and sterilization timedata in accordance with certain aspects of the present invention.

DETAILED DESCRIPTION

Aspects of the present invention overcome the above identified problems,as well as others, by providing systems, methods, and devices forsterilizing an antiseptic solution while maintaining antimicrobialefficacy and while complying with regulatory requirements.

Various aspects of an antiseptic applicator may be illustrated withreference to one or more exemplary embodiments. As used herein, the term“exemplary” means “serving as an example, instance, or illustration,”and should not necessarily be construed as preferred or advantageousover other embodiments of sterilization methods disclosed herein.

The term “about” as used herein preferably means ±5% and more preferably±1% of the provided value.

Aspects of the present invention include a method of sterilizingantiseptic solution contained in a container. The method may includeheating antiseptic solution contained within a container or ampoule to acertain temperature and maintaining the temperature for a certain amountof time sufficient to sterilize the solution while maintainingsufficient purity of the antiseptic solution to comply with regulatoryrequirements. The antimicrobial efficacy directly relates to the purityof the antiseptic solution. Generally, when the purity of the antisepticmolecules is too low, the solution is not as effective as anantimicrobial solution. Furthermore, higher levels of impurities withinan antiseptic solution can have a deleterious impact on patient health.

The container is preferably a self-contained structure, formed of amaterial suitable for containing the antiseptic solution. In an aspect,the container may be made of a frangible material such that uponapplication of sufficient force the container fractures. For example,the material may comprise plastic or glass. The terms “container” and“ampoule” are used interchangeably herein. The wall of the container mayhave a thickness sufficient to withstand the sterilization process,transport, and storage. When the container is frangible, the materialand thickness may also be sufficient to allow the container to befractured upon the application of localized pressure. The thicknessrange may vary depending on the container size. Example thicknessesinclude from about 0.15 mm to about 0.45 mm. In another example aspect,the container may comprise a non-frangible material, such as a metalsuch as steel, aluminum, etc., capable of withstanding the sterilizationprocess.

While antiseptic solutions are of particular focus herein, the containermay alternatively contain medicaments, chemical compositions, cleansingagents, cosmetics, or the like. For example, the container may be filledwith antiseptic compositions (e.g., compositions comprising one or moreantiseptic molecules), preferably an antimicrobial liquid or gelcomposition.

The antiseptic solution may comprise an alcoholic solvent. For example,the alcoholic solvent may be selected from the group consisting ofethanol, isopropanol, and n-propanol. The amount of solvent may be fromabout 40% v/v to about 90% v/v, more preferably about 50% v/v to about80% v/v, and still more preferably about 65% v/v to about 80% v/v. Theremaining volume of the solution may be water or another solvent. Forexample the solution may contain from about 10% v/v to about 60% v/v,more preferably about 20% to about 50% v/v, and still more preferablyabout 20% to about 35% v/v water.

The container may contain antiseptic solution of a sufficient amount,sufficient concentration, and sufficient purity to be applied to adesired surface and have an antimicrobial effect on the desired surface.In one aspect, the desired surface is a patient's skin. It will beappreciated that the amount of antiseptic solution may vary. In oneaspect the amount of antiseptic solution may be 0.01-100 mL ofantiseptic. More preferably, the amount of antiseptic solution neededmay be about 0.5-60 mL and still preferably may be about 0.5-30 mL.Examples include 0.67, 1, 1.5, 3, 10.5, and 26 mL of antiseptic.

Suitable antiseptic molecules include bis-(dihydropyridinyl)-decanederivatives, octenidine salts, cationic surfactants, biguanides, andgenerally cationic antiseptic molecules. Preferred antiseptic agentsinclude octenidine, such as octenidine dihydrochloride, andchlorhexidine, such as chlorhexidine gluconate. The concentration of theantiseptic may vary depending on the specific antiseptic species used orthe desired antimicrobial effect that is desired. For example, whenusing octenidine or an octenidine salt the concentration may vary fromabout 0.0001% w/v to about 2.0% w/v, more preferably from about 0.01%w/v to about 0.5% w/v, and still more preferably from about 0.1% w/v toabout 0.4% w/v. When chlorhexidine or a chlorhexidine salt is used, theconcentration may be from about 0.1% w/v to about 2.5% w/v, morepreferably from about 0.5% w/v to about 2.25% w/v, and still morepreferably about 1.2% w/v to about 2.0% w/v.

In an aspect, when chlorhexidine or a chlorhexidine salt is used, thepurity of the solution, when applied to the skin (e.g., after thesterilization method described herein), may be at least about 92% pure,more preferably at least about 94% pure, still more preferably at leastabout 96% pure, and still more preferably at least about 98% pure. Asused herein, purity means the percent concentration of antisepticmolecules in solution relative to the total concentration of antisepticmolecules plus concentration of substances that are derived from orrelated to the antiseptic molecule. For example, a 95% pure antisepticsolution means that if there are 100 molecules that are eitherantiseptic molecules or molecules derived from or related to theantiseptic molecule, 95 of the molecules are the antiseptic molecule and5 of those molecules are derived from or related to the antisepticmolecule. These molecules derived from or relating to the antisepticmolecule have reduced or no antimicrobial activity. Thus, a lower puritysolution will have lower antimicrobial efficacy as fewer of the targetantiseptic molecules are delivered to the patient's skin. Further, alower purity solution will not comply with regulatory requirements. Bymeasuring the concentration of antiseptic molecules in solution ascompared to concentration of antiseptic molecules and molecules derivedfrom or related to the antiseptic molecule, one can determine the purityof the solution and whether the purity is sufficient to comply withregulatory requirements.

In a preferred aspect, the antiseptic solution provided in the containercomprises about 70% v/v alcohol solvent in water and about 2.0% w/vantiseptic molecules. In a preferred aspect the solvent may beisopropanol and the antiseptic molecule may be chlorhexidine gluconate.

It has been found that when the antiseptic solution within the containeris brought to a particular temperature and maintained at thattemperature for a particular amount of time, the solution issufficiently sterilized while maintaining sufficient antimicrobialefficacy as an antiseptic and while satisfying regulatory requirements.In an aspect of the present invention, the antiseptic solution may bebrought to a temperature (also referred to herein as the “sterilizationtemperature”) from about 85° C. to about 135° C., more preferably about90° C. to about 125° C., and still more preferably about 95° C. to about120° C.

As used herein, the term “sterilization time” means the length of timeat which the solution is at the sterilization temperature. That is, the“sterilization time” does not include the time it takes for a solutionto reach the sterilization temperature (i.e., does not include “ramp up”time) and also does not include the time it takes for the solution toreturn to the temperature the solution was at prior to the heating(i.e., does not include “cool down” time). The time it takes for thetemperature of the solution to reach the sterilization temperature isreferred herein as the “ramp up” time and the time to return to thestarting temperature is referred herein as the “cool down” time. As usedherein, the term “sterilization temperature” means the temperature ortemperature range that the solution reaches and maintains during thesterilization time, independent of the starting temperature of thesolution. For purposes of illustration only, a sterilization time of 90minutes and a sterilization temperature of 95° C. for a solutionstarting at 21° C. would mean that the period of time starting from themoment the solution reaches 95° C. and ending the moment the solutionfalls below 95° C. during the beginning of the cool down process is 90minutes. Thus, the time it takes from the solution to rise from 21° C.to 95° C. (i.e., ramp-up time) and the time it takes for the solution toreturn to 21° C. (i.e., cool-down time) is not included in thesterilization time.

In an aspect, the sterilization time may be no more than about 19 hours,more preferably no more than about 13 hours, more preferably no morethan about 5 hours, more preferably no more than about 3 hours, morepreferably no more than 2 hours, more preferably no more than 1 hour,more preferably no more than 40 minutes, more preferably no more thanabout 25 minutes, more preferably no more than 6 minutes, and morepreferably no more than 1 minute.

It has been found that combinations of sterilization temperature andsterilization time can be selected to provide a sterilized antisepticsolution having sufficient purity to comply with regulatory requirementswhen used as an antiseptic. For example, for a sterilization temperatureof about 85° C., the sterilization time may be from about 9 hours toabout 19 hours. For a sterilization temperature of about 95° C., thesterilization time may be at least about 1.5 hours and up to about 6.5hours. For a sterilization temperature of about 105° C., thesterilization time may be from about 17 minutes to about 2.5 hours. Fora sterilization temperature of about 110° C., the sterilization time maybe at least about 6 minutes and up to about 90 minutes. For asterilization temperature of about 115° C., the sterilization time maybe from about 3 minutes to about 55 minutes. For a sterilizationtemperature of about 120° C., the sterilization time may be at leastabout 2 minutes and up to about 35 minutes. For a sterilizationtemperature of about 125° C., the sterilization time may be from about 1minute to about 22 minutes. For a sterilization temperature of about130° C., the sterilization time may be at least about 1 minute and up to14 minutes. For a sterilization temperature of about 135° C., thesterilization time may be from about 1 minute to about 9 minutes. In anaspect of the present invention, the above example sterilizationtemperatures and sterilization times may be applied to an antisepticsolution comprising about 70% v/v isopropanol and about 2.0% w/vchlorhexidine gluconate or other antiseptic solutions described above.

It has been found that heating the antiseptic solution contained in thecontainer to the above sterilization temperatures and maintaining thetemperature for the above sterilization times, sufficiently sterilizesthe solution, while maintaining sufficient purity to comply withregulatory requirements. The amount of degradation of the antisepticmolecule can be quantified by measuring the initial purity of antisepticsolution prior to the ramp up time (i.e., prior to the process ofbringing the solution up to the sterilization temperature) and measuringthe post-sterilized purity of antiseptic solution after the cool downtime (i.e., after the antiseptic solution returns to the temperature thesolution was at prior to the process of bringing the solution up to thesterilization temperature). Thus, as used herein, the “initial purity”is the purity prior to ramp up and “post-sterilization purity” is thepurity of the solution after cool down. In an aspect of the presentinvention, the initial purity of the antiseptic solution, e.g.,chlorhexidine gluconate, may be at least about 94%, preferably at leastabout 97%, and more preferably at least about 98%. The meaning of purityis provided above. The resulting post-sterilized solution is found tohave sufficient purity to provide the desired antimicrobial efficacy asan antiseptic and to comply with regulatory requirements.

In an example aspect, it has been found that chlorhexidine gluconatemolecules degrade into one or more the following molecules when heattreated:N-[[6-[[[(4-chlorophenyl)carbamimidoyl]carbamimidoyl]-amino]hexyl]carbamimidoyl]urea,N-(4-chlorophenyl)guanidine, N-(4-chlorophenyl)urea,1-(6-aminohexyl)-5-(4-chlorophenyl) biguanide,N-(4-chlorophenyl)-N′-[[6-[[[(4-chlorophenyl)carbamimidoyl]carbamimidoyl]amino]hexyl]carbamimidoyl]urea,1-(4-chlorophenyl)-5-[6-[[(phenylcarbamimidoyl)carbamimidoyl]amino]hexyl]biguanide,1-[6-(carbamimidoylamino)hexyl]-5-(4-chlorophenyl)-biguanide, and4-chloroaniline. Thus, in an example aspect, the purity of the solutioncan be determined by comparing the amount of chlorhexidine to all of theabove-listed chlorhexidine gluconate related substances. However, itshould be noted that the above list is not exhaustive. One havingordinary skill in the art would be able to determine which molecules aredegredants of the antiseptic molecule after the sterilization process.

As noted above, the purity of the antiseptic solution after the heatinghas been terminated and when the solution has returned to thetemperature the solution was at prior to the process of bringing thesolution up to the sterilization temperature (for example ambienttemperature) is referred herein as the post-sterilization purity. Asnoted above, the post-sterilization purity is preferably measured whenthe antiseptic solution has cooled because degradation may occur duringcooling. In an aspect of the present invention, by selecting anappropriate combination of sterilization temperature and sterilizationtime, the post-sterilization purity may be maintained relatively closeto the initial purity, while still being sterile. In particular, thecombination of sterilization temperature and sterilization time arechosen such that the percentage point change in purity from the initialpurity to the post-sterilization purity is at most about 5%, morepreferably at most about 4%, more preferably at most about 3%, and mostpreferably at most about 2%. It should be understood that the percentagepoint change refers to the absolute percentage point difference betweenthe initial purity and the post-sterilization purity. For example, achange in initial purity of 95% to a post-sterilization purity of 90% isa percentage point change of 5%.

In addition to maintaining a sufficient purity, it has been found thatthe proper combination of sterilization temperature and sterilizationtime can be selected such that the solution is sterile. As used herein,sterile means “7 day sterility” as tested following the proceduresdescribed in U.S. Pharmacopeial Convention (USP) Chapter 55 “BiologicalIndicators—Resistance Performance Tests,” USP 36; Official from May 1,2013. Sterile also means completely free of microbes, immediatelyfollowing sterilization. In an aspect, Geobacillus stearothermophilusmay be used as a test microbe. Thus, in an aspect, a sterile solutionwould have no growth of Geobacillus stearothermophilus shown by the ‘7day sterility’ testing described above. In another aspect, a solutioninoculated with Geobacillus stearothermophilus would be completely freeof viable Geobacillus stearothermophilus immediately following thesterilization method.

In another aspect of the present invention, it was found that theinventive method has a sterility assurance level (SAL) of at least about10⁻⁶ under particular combination of sterilization temperature andsterilization time. SAL is a measurement of probability of amicroorganism occurring on an item following a sterilization procedure.A SAL of 10⁻⁶ means there is a 1 in 1,000,000 chance of a viablemicroorganism occurring in a sterilized product. Thus, the SAL measuresthe probability of a sterilization method resulting in a non-sterilizedproduct. The calculation to determine SAL is described in more detail inthe below examples. For example, it has been found that a method ofexposing the antiseptic solution to a temperature of 100° C. for about50 minutes, a temperature of 105° C. for about 17 minutes, or 110° C.for about 6 minutes would each have a SAL of at least 10⁻⁶ (i.e., a1/1,000,000 chance that a viable microbe will be present in a sterilizedsolution).

As noted above, after the sterilization time ends, the solution may becooled. For example, it may take about 10 to about 40 minutes to coolthe antiseptic solution following the sterilization time. The time canbe shortened using a cooling device. This additional time correlateswith the particular sterilization temperature. For examples, a highersterilization temperature (e.g., 125° C.) as compared to a lowersterilization (e.g., 85° C.) would take longer to return to roomtemperature after sterilization. Thus, the overall processing time,including cool down, may include an additional about 10 to about 20minutes longer than the sterilization time.

It is within the scope of the invention that any machine capable ofheating the antiseptic solution to the sterilization temperature andmaintaining the solution at the sterilization temperature for thesterilization time may be used, while preferably limiting the ramp uptime. Example equipment that may include a cascading water sterilizer.When using the cascading water sterilizer the ramp up time may be about15 minutes, while the cool down time may be about 25 minutes. Thecascading water sterilizer provides a constant stream of water whichheats the solution to the sterilization temperature, maintains thesterilization temperature over the entirety of the sterilization time,and finally cools the solution.

As provided above, example combinations of sterilization time andsterilization temperature that provide a sterilized solution withsufficient purity to satisfy regulatory requirement are as follows. Fora sterilization temperature of about 85° C., the sterilization time maybe from about 9 hours to about 19 hours. For a sterilization temperatureof about 95° C., the sterilization time may be at least about 1.5 hoursand up to about 6.5 hours. For a sterilization temperature of about 105°C., the sterilization time may be from about 17 minutes to about 2.5hours. For a sterilization temperature of about 110° C., thesterilization time may be at least about 6 minutes and up to about 90minutes. For a sterilization temperature of about 115° C., thesterilization time may be from about 3 minutes to about 55 minutes. Fora sterilization temperature of about 120° C., the sterilization time maybe at least about 2 minutes and up to about 35 minutes. For asterilization temperature of about 125° C., the sterilization time maybe from about 1 minute to about 22 minutes. For a sterilizationtemperature of about 130° C., the sterilization time may be at leastabout 1 minute and up to 14 minutes. For a sterilization temperature ofabout 135° C., the sterilization time may be from about 1 minute toabout 9 minutes.

EXAMPLES

A sample of antiseptic solution of 70% v/v isopropanol, 30% v/v water,and 2.0% w/v chlorhexidine gluconate contained in a glass ampoule wastested in each of the following examples. An inoculum of greater than1,000,000 but less than 10,000,000 test spores of Geobacillusstearothermophilus were inserted and sealed into the container. In thefollowing examples, a 10 mL sample of antiseptic solution at roomtemperature was placed in either a water or oil bath (water bath fortemperatures of ≤95′C; oil bath for temperatures≥100° C.) having apreset temperature (i.e., the sterilization temperature). The ampoulecontaining chlorhexidine gluconate solution and test spores was placedin the heating medium. The sample with test spores was removed at aparticular time (i.e., the sterilization time), allowed to cool to roomtemperature, then tested and incubated over a seven day period forbacterial growth. Samples of antiseptic solution also stored at thepreset temperature were tested for degradation of chlorhexidinegluconate. The 7-day bacterial growth testing followed the proceduresdescribed in U.S. Pharmacopeial Convention (USP) Chapter 55 “BiologicalIndicators—Resistance Performance Tests,” USP 36; Official from May 1,2013. Tables 1-6 show the data collected for purity and sterility of achlorhexidine gluconate solution that was 98.67% pure prior to the heattreatment. The purity percent values listed in the tables are theabsolute purity of the chlorhexidine gluconate after heat treatment andcooling to ambient temperature. The Δpurity percent values are thepercentage point change relative to the baseline purity. For example, inTable 1, at 78° C. and 4 hours the purity of chlorhexidine gluconate was98.05%, which is a 0.62% percentage point change from the initial purityof 98.67%.

TABLE 1 78° C., Initial Purity 98.67%, water bath Time (hours) 4 6 816-18 24 48 Purity (%) 98.05% 97.86% 97.61% 96.70% 95.85% 92.84% ΔPurity 0.62% 0.81%  1.05%  1.97% 2.82%  5.82% (%) Sterility Not sterile Notsterile Not sterile Sterile 7 days (days) Growth at 1 Growth at 2 Growthat 2 day days days

TABLE 2 80° C., Initial Purity 98.67%, water bath Time (hours) 6 8 1016-18 24 48 Purity 97.85% 97.50% 97.26% 96.49% 95.14% 91.55% (%) ΔPurity0.81% 1.17% 1.41% 2.18% 3.53% 7.12% (%)

TABLE 3 82° C., Initial Purity 98.67%, water bath Time (hours) 4 6 8 1016-18 24 48 Purity 97.72% 97.38% 97.03% 96.73% 95.70% 94.31% 89.63% (%)ΔPurity  0.94% 1.29%  1.64% 1.94%  2.97% 4.35%  9.03% (%) Sterility Notsterile Not sterile Sterile 7 Sterile 7 days (days) Growth at Growth atdays 1 day 1 day

TABLE 4 85° C., Initial Purity 98.67%, water bath Time (hours) 2 4 6 810 16-18 24 48 Purity 98.11% 97.46% 97.09% 96.60% 96.21% 93.96% 92.11%85.54% (%) ΔPurity  0.56% 1.20% 1.58%  2.07% 2.46%  4.70% 6.56% 13.13%(%) Sterility Not Sterile 7 Sterile 7 Sterile 7 (days) sterile days daysdays Growth at 1 day

TABLE 5 90° C., Initial Purity 98.67%, water bath Time (hours) 2 4 6 810 16-18 24 48 Purity 97.50% 96.66% 95.79% 94.88% 94.37% 91.59% 87.51%73.67% (%) ΔPurity 1.17% 2.01% 2.88% 3.79% 4.30% 7.08% 11.16% 25.00% (%)

TABLE 6 95° C., Initial Purity 98.67%, water bath Time (hours) 0.25 0.51.25 2 4 6 8 10 16-18 24 48 Purity 97.05% 95.53% 94.74% 92.77% 91.04%77.97% 49.72% (%) ΔPurity 1.62%  3.14% 3.93%  5.89% 7.63% 20.70% 48.95%(%) Sterility Not Not Sterile Sterile 7 Sterile 7 Sterile Sterile 7(days) sterile sterile 7 days days days 7 days days Growth Growth at 1at 4 day day

Additional experiments were performed in an oil bath to test puritychanges at 105° C. and 115° C. Glass ampoules containing the antisepticsolution were subjected to the sterilization times and sterilizationtemperatures shown in Table 7 and Table 8 using an oil bath. The changein % purity for the antiseptic solution after the sterilization time wasmeasured and compared to the initial % purity values. The measurementswere taken after the solution returned to ambient temperature.

TABLE 7 105° C. Initial Purity 98.7%, oil bath Time (hours) 0.25 0.50.833 1 1.5 Purity (%) 98.3% 98.0% 97.2% 96.9% 95.7% ΔPurity (%) 0.400.80 1.58 1.89 3.03

TABLE 8 115° C., Initial Purity 98.7% oil bath Time (hours) 0.1667 0.3330.5 0.667 0.833 Purity (%) 97.9% 97.1% 96.2% 95.2 94.3% ΔPurity (%) 0.821.61 2.59 3.54 4.50

The above data was then used to prepare an Arrhenius equation using thestandard method in the art. The use of an Arrhenius equation is awell-known and accepted method of modeling temperature dependence onreaction rate. Using the Arrhenius equation, the following predictedvalues for purity were obtained:

TABLE 9 Predicted Purity Using Arrhenius Equation Sterilization MaximumSterilization Time to maintain a Temperature specified purity change(min) (° C.) 2% 3% 4% 5% 85 N/A 670 900 1123 90 260 390 525 654 95 155230 310 386 100 92 140 185 231 105 55 85 113 140 110 35 52 70 86 115 2232 43 54 120 14 20 27 34 125 9 13 17.5 21.5 130 5.5 8.5 11 14 135 3.75.5 7.3 9.1

The measured impact of various sterilization temperatures andsterilization times on the characteristics of the antiseptic are shownbelow. Table 10 summarizes the change in % purity for the chlorhexidinegluconate after exposure to various sterilization temperatures andsterilization times. The percent change in purity is made by comparingthe purity of solution prior to the ramp up time (i.e., prior to theprocess of bringing the solution up to the sterilization temperature)with the purity of solution after the cool down time (i.e., after thesolution returns ambient temperature). ‘W’ indicates that thesterilization temperature and sterilization time would result in thechange of purity of not more than 2%. The ‘X’, ‘Y’ and ‘Z’ indicate thatthe sterilization temperature and sterilization time would result in achange of purity of not more than 3%, 4% and 5%, respectively. Finally,an ‘A’ indicates that the sterilization temperature and sterilizationtime would result in the change of purity of greater than 5%.

TABLE 10 Effect of Heat and Temperature on Chemical StabilitySterilization Time (hours) 0.17 0.25 0.33 0.5 0.67 0.83 1 1.5 2 4 6 8 1016-18 24 Sterilization 78 W W W W X Temp (° C.) 80 W W W X Y 82 W W W WX Z 85 W W W X X Z A 90 W X X Y Z A A 95 W Y Y A A A 105 W W W W Y 115 WW X Y Z KEY: W = the solution had a change in purity of not more than 2%X = the solution had a change in purity of not more than 3% Y = thesolution had a change in purity of not more than 4% Z = the solution hada change in purity of not more than 5% A = the solution had a change inpurity above 5%

Table 11 summarizes the measured sterility of an antiseptic solutioncontaining greater than or equal to 1,000,000 but less than 10,000,000test spores of Geobacillus stearothermophilus after exposure to varioussterilization temperatures sterilization times.

TABLE 11 Effect of Heat and Temperature on Sterility Sterilization Time(hours) 0.05 0.1 0.25 0.4 0.5 0.7 1.25 4 6 8 18 24 48 Sterilization 78 NN N Y Temp (° C.) 82 N N Y Y 85 N Y Y Y 95 N N Y Y Y Y Y 100 N N N Y 105N Y 110 Y Y KEY: Y = the solution was sterile as shown by absence ofviable bacterial spores N = the solution was not sterile as evidenced bygrowth of viable bacterial spores

As shown in the above tables when varying sterilization time andtemperature, there is a particular window where the treated antisepticsolution is sterile and has change in purity of less than a certainpercentage, for example 5%. It should be noted that because the abovedata is a threshold analysis, one can extrapolate the results for othersterilization times. Once it is found that the change in purity is atleast 5% at particular temperature and time, it can be presumed thatlonger sterilization times at the same temperature will further degradethe solution. It can also be presumed that all samples starting with thesame initial purity value which are sterilized for shorter times at thesame temperature below the time found to have lower than a 5% change inpurity will also have change in purity lower than 5%. For example, thesample tested at 95° C. for 4 hours had 95.53% purity, while the sampletested at 95° C. for 6 hours had 94.74%. Accordingly, it can beextrapolated that all sterilization times greater than 6 hours at 95° C.will have less than 95.74% purity, while all sterilization times lessthan 4 hours at 95° C. will have at least 95.53% purity. Similarly, withrespect to the USP seven-day sterility test, once it is found that asample has seven-day sterility at a particular sterilization temperatureand time, it can be presumed that longer sterilization time at the sametemperature will also exhibit seven day sterility (i.e., long termsterility). Thus, it can presumed that all samples sterilized for longertimes at the same temperature beyond the time found to have seven-daysterility will also have seven day sterility. It can also be presumedthat all samples sterilized at shorter times but at the same temperaturecompared to a sample found not to have seven-day sterility will also nothave seven day sterility. For example, the sample tested at 95° C. for1.25 hours was sterile after seven days, while the sample tested at 95°C. for 0.5 hours had bacterial growth within four days (i.e., did nothave seven day sterility). Accordingly, it can be extrapolated that allsterilization times greater than 1.25 hours at 95° C. will be sterileafter seven days while all sterilization times less than 0.5 hours at95° C. will have bacterial growth within seven days.

The same can be done for other threshold values (e.g., changes in puritybelow or higher than 5%, such as 2%, 3%, and 4%).

In addition to above testing, further testing was conducted to determineat what time the Sterility Assurance Level (SAL) of 10⁻⁶ can be reachedat a certain temperature. The USP 55 “Biological Indicators—ResistancePerformance Tests” procedures were followed to determine the SAL.Greater than or equal to 1,000,000 test spores of Geobacillusstearothermophilus, but less than 10,000,000, were inserted into a 1 mLsample of antiseptic solution comprising 70% v/v isopropanol in waterand 2.0% w/v chlorhexidine gluconate. The samples were tested at 100°C., 105° C., and 110° C. for various times. Ten samples were tested ateach time point. The results were as follows:

TABLE 12 Sterility Test Results for SAL Testing 100° C. 105° C. 110° C.Time Result Time Result Time Result (min) (# positive) (min) (#positive) (min) (# positive) 10 10 6 10 3 10 15 9 7.5 5 3.5 0 20 3 9 2 41 25 3 10 0 4.5 0 30 2 12 2 5 0 35 3 14 0 6 0 40 0 16 0 7 0 50 0 20 0 80 24 0 10 0 12 0 14 0

The above results are expressed as the number of positives recorded outof ten samples tested. For example, “10” means 10 samples out of 10samples tested positive for microbes (non-sterile). The above data wasthen used to calculate the “D-values,” in accordance with USP 55procedures. The term D-value has the normal meaning as used inmicrobiology. Specifically, it refers to decimal reduction time and isthe time required at a certain temperature to kill 90% of the organismsbeing studied. Thus after a colony is reduced by 1 D, only 10% of theoriginal organisms remain, i.e., the population number has been reducedby one decimal place in the counting scheme. D-values can be calculatedusing the Holcomb-Spearman-Karber Method (HSK), which is a data analysisknown in the art (see USP 55 procedures and Block, Seymour S.“Disinfection, Sterilization, and Preservation.” Philadelphia, Pa.:Lippincott Williams & Wilkens, 120-122. 2001). Applying the HSK methodto the above Table 12 data, the resulting D-values were calculated alongwith upper and lower confidence limits:

TABLE 13 D-Values Lower Confidence Upper Confidence Temp (° C.) D-value(min) 95% (min) 95% (min) 84 51 44 59 100 3.55 3.06 4.05 105 1.29 1.161.41 110 0.50 0.48 0.51

The D-values can be used to calculate a sterility assurance Level (SAL)(see USP 55 procedures). SAL is a term used in microbiology to describethe probability of a single unit being non-sterile after it has beensubjected to a sterilization process. A 10⁻⁶ SAL means there is a1/1,000,000 chance that a single viable microbe will remain insterilized items. By extrapolating the log reduction rates following anextreme, artificially high initial contamination, the sterilizationprocedure must encompass 12 log increments (multiplying the D-value by12), overkill conditions, in order to verify an SAL of 10. Erring on theside of caution, the upper confidence limit D-values were used tocalculate the following time to achieve 10⁻⁶ SAL:

TABLE 14 SAL 10⁻⁶ Time Time to achieve SAL 10⁻⁶ Temp (° C.) (minutes) 84612.00 100 48.58 105 16.97 110 6.17

Thus, as indicated in Table 14, exposing the antiseptic solution to atemperature of 100° C. for about 48.58 minutes, a temperature of 105° C.for about 16.97 minutes, or 110° C. for about 6.17 minutes would eachhave a SAL of 10⁻⁶ (i.e., a 1/1,000,000 chance that a viable microbewill be present following the sterilization process).

Using standard mathematical modeling, the above four D-value data pointsfrom Table 14 were used to prepare a exponential predictive functionhaving the following formula:y=1,553,000,000·e ^((−0.1747) x)  (I)where y is time in minutes and x is temperature in degrees Celsius.Thus, Formula (I) indicates at a given temperature the minimum time forachieving at least a 10⁻⁶ SAL. Using Formula (I), the followingpredictive data points were generated:

TABLE 15 Predictive SAL 10⁻⁶ Time Time to achieve SAL 10⁻⁶ Temp (° C.)(min) 85 552.23 95 96.25 115 2.92 120 1.22 125 0.51 130 0.21 135 0.09The times found in Tables 14 and 15 have been rounded as follows inTable 16:

TABLE 16 Rounded SAL 10⁻⁶ Time Time to achieve SAL 10⁻⁶ Temp (° C.)(min) 85 552 95 96 100 48 105 17 110 6.2 115 3 120 2 125 1 130 1 135 1

The rounded data points are plotted in FIG. 1. FIG. 1 illustrates thesterilization times and temperatures fit to functions which capture theparameter space (time and temperature) to maintain a specific change inpurity following the sterilization process (area between curves). Thedata points in FIG. 1 include data points from Table 9 and Table 16above. The black squares represent the data points from 85° C. to 120°C. where the corresponding times were sterile. The grey squaresrepresent data points from 125° C. to 135° C. where the correspondingtimes were sterile. The following natural log formula was fitted to thesquare data points from 85° C. to 125° C.:y=−6.14·ln x+123.2 for 1≤x≤552  (II)where y is the temperature in degrees Celsius and x is the time inminutes. From 125° C. to 135° C., the time x is a constant 1 minute.

The data points found in Table 9 above were also plotted in FIG. 1. Theblack diamonds represent the data points from 85° C. to 135° C. wherethe corresponding times had at most a percent change in purity of 5%.The black triangles represent the data points from 85° C. to 135° C.where the corresponding times had at most a percent change in purity of4%. The black circles represent data points from 85° C. to 135° C. wherethe corresponding times had at most a % change in purity of 3%. The greytriangles represent data points from 90° C. to 135° C. where thecorresponding times had at most a percent change in purity of 2%. Therewas no time at 85° C. where the solution would have at most a percentchange in purity of 2%, The following natural log formula was fitted tothe black diamond data points (i.e., the points having at most 5% changein purity):y=−10.38·ln x+156.9 for 9.1≤x≤1123  (III)

The following natural log formula was fitted to the black triangle datapoints (i.e., the points having at most 4% change in purity):y=−10.37·ln x+154.6 for 7.3≤x≤900  (IV)where y is the temperature in degrees Celsius and x is the time inminutes. The following natural log formula was fitted to the blackcircle data points (i.e., the points having at most 3% change inpurity):y=−10.4·ln x+151.7 for 5.5≤x≤670  (V)where y is the temperature in degrees Celsius and x is the time inminutes. The following natural log formula was fitted to the greytriangle data points (i.e., the points having at most 2% change inpurity):y=−10.6·ln x+148.3 for 3.7≤x≤260  (VI)where y is the temperature in degrees Celsius and x is the time inminutes.

As can be seen in FIG. 1, the area above Formula (II) but below formula(III), within the temperature range of 85° C. to 125° C., representstemperature and time combinations that provide a sterile solution withat most a 5% change in purity. This area can thus be presented by thefollowing relationship:y≥−6.14·ln x+123.2 for 1≤x≤552andy≤−10.38·ln x+156.9 for 21.5≤x≤1123,where y is temperature in degrees Celsius and x is time in minutes.Similarly, within the temperature range of 125° C. to 135° C., the areaabove the constant line x=1 and below formula (III) representstemperature and time combination that provide a sterile solution with amost 5% change in purity. This area can be presented by the followingrelationship:x≥1andy≤−10.38·ln x+156.9 for 9.1≤x≤21.5where y is the temperature in degrees Celsius and x is time in minutes.

As can be seen in FIG. 1, the area above Formula (II) but below formula(IV), within the temperature range of 85° C. to 125° C., representstemperature and time combinations that provide a sterile solution withat most a 4% change in purity. This area can thus be presented by thefollowing relationship:y≥−6.14·ln x+123.2 for 1≤x≤552andy≤−10.37·ln x+154.6 for 17.5≤x≤900,where y is temperature in degrees Celsius and x is time in minutes.Similarly, within the temperature range of 125° C. to 135° C., the areaabove the constant line x=1 and below formula (IV) representstemperature and time combination that provide a sterile solution with amost 4% change in purity. This area can be presented by the followingrelationship:x≥1andy≤−10.37·ln x+154.6 for 7.3≤x≤17.5where y is the temperature in degrees Celsius and x is time in minutes.

As can be seen in FIG. 1, the area above Formula (II) but below formula(V), within the temperature range of 85° C. to 125° C., representstemperature and time combinations that provide a sterile solution withat most a 3% change in purity. This area can thus be presented by thefollowing relationship:y≥−6.14·ln x+123.2 for 1≤x≤552andy≤−10.4·ln x+151.7 for 13≤x≤670where y is temperature in degrees Celsius and x is time in minutes.Similarly, within the temperature range of 125° C. to 135° C., the areaabove the constant line x=1 and below formula (V) represents temperatureand time combination that provide a sterile solution with a most 3%change in purity. This area can be presented by the followingrelationship:x≥1andy≤−10.4·ln x+151.7 for 5.5≤x≤13,where y is the temperature in degrees Celsius and x is time in minutes.

As can be seen in FIG. 1, the area above Formula (II) but below formula(VI), within the temperature range of 90° C. to 125° C., representstemperature and time combinations that provide a sterile solution withat most a 2% change in purity. This area can thus be presented by thefollowing relationship:y≥−6.14·ln x+123.2 for 1≤x≤552andy≤−10.6·ln x+148.3 for 9≤x≤260where y is temperature in degrees Celsius and x is time in minutes.Similarly, within the temperature range of 125° C. to 135° C., the areaabove the constant line x=1 and below formula (VI) representstemperature and time combination that provide a sterile solution with amost 2% change in purity. This area can be presented by the followingrelationship:x≥1andy≤−10.6·ln x+148.3 3.7≤x≤9where y is the temperature in degrees Celsius and x is time in minutes.

While aspects of the present invention have been described in connectionwith illustrative implementations, it will be understood by thoseskilled in the art that variations and modifications of the aspectsdescribed above may be made without departing from the scope hereof.Other variations will be apparent to those skilled in the art from aconsideration of the specification or from a practice along the lines asdisclosed herein.

The invention claimed is:
 1. A sterile antiseptic solution comprising:an antimicrobial molecule selected from the group consisting of: anoctenidine salt and a biguanide; and an alcohol solvent, wherein thesterile antiseptic solution is sterile and at least about 92% pure,wherein the sterile antiseptic solution is made sterile by a methodcomprising the steps of: providing a container containing an antisepticsolution, the antiseptic solution having an initial purity; selecting asterilization temperature from about 85° C. to about 135° C. and asterilization time from about 1 minute to about 19 hours; heating theantiseptic solution to the selected sterilization temperature;maintaining the antiseptic solution at the sterilization temperature forthe selected sterilization time; and terminating the heating of theantiseptic solution when the selected sterilization time expires,thereby providing the sterile antiseptic solution, wherein afterterminating the heating, the sterile antiseptic solution has apost-sterilization purity, and wherein the sterilization temperature andthe sterilization time are selected such that after terminating theheating, the sterile antiseptic solution has a post-sterilization purityof at least about 92% and a percentage point change in purity from theinitial purity to the post-sterilization purity of at most about 5%. 2.The sterile antiseptic solution of claim 1, wherein the selectedsterilization time and selected sterilization temperature satisfy thefollowing relationship: a) 85° C.≤y<125° C., y≥−6.14·ln x+123.2 for1≤x≤552, and y≤−10.38·ln x+156.9 for 21.5≤x≤1123; or b) 125° C.≤y≤135°C., x≥1 and y≤−10.38·ln x+156.9 for 9.1≤x≤21.5, wherein y is thesterilization temperature and x is the sterilization time in minutes. 3.The sterile antiseptic solution of claim 1, wherein the selectedsterilization time and selected sterilization temperature satisfy thefollowing relationship: a) 85° C.≤y<125° C., y≥−6.14·ln x+123.2 for1≤x≤552, and y≤−10.37·ln x+154.6 for 17.5≤x≤900; or b) 125° C.≤y≤135°C., x≥1 and y≤−10.37·ln x+154.6 for 7.3≤x≤17.5, wherein y is thesterilization temperature and x is the sterilization time in minutes. 4.The sterile antiseptic solution of claim 1, wherein the selectedsterilization time and selected sterilization temperature satisfy thefollowing relationship: a) 85° C.≤y<125° C., y≥−6.14·ln x+123.2 for1≤x≤552, and y≤−10.4·ln x+151.7 for 13≤x≤670; or b) 125° C.≤y≤135° C.,x≥1 and y≤−10.4·ln x+151.7 for 5.5≤x≤13, wherein y is the sterilizationtemperature and x is the sterilization time in minutes.
 5. The sterileantiseptic solution of claim 1, wherein the selected sterilization timeand selected sterilization temperature satisfy the followingrelationship: a) 90° C.≤y<125° C., y≥−6.14·ln x+123.2 for 1≤x≤552, andy≤−10.6·ln x+148.3 for 9≤x≤260; or b) 125° C.≤y≤135° C., x≥1 andy≤−10.6·ln x+148.3 for 3.7≤x≤9, wherein y is the sterilizationtemperature and x is the sterilization time in minutes.
 6. The sterileantiseptic solution of claim 1, wherein the sterile antiseptic solutionis completely free of microbes.
 7. The sterile antiseptic solution ofclaim 1, wherein the antimicrobial molecule is selected from the groupconsisting of chlorhexidine gluconate and octenidine dihydrochloride;and wherein the solvent is selected from the group consisting ofethanol, isopropanol, and n-propanol.
 8. The sterile antiseptic solutionof claim 1, comprising from about 40% to about 90% v/v of the solventand from about 0.1% to about 2.5% w/v of the antimicrobial molecule,wherein the solvent is isopropanol and the antimicrobial molecule ischlorhexidine gluconate.
 9. The sterile antiseptic solution of claim 1,comprising about 70% v/v of the solvent and about 2.0% w/v of theantimicrobial molecule, wherein the solvent is isopropanol and theantimicrobial molecule is chlorhexidine gluconate.
 10. The sterileantiseptic solution of claim 1, wherein the sterilization time is fromabout 6 minutes to about one hour.
 11. The sterile antiseptic solutionof claim 1, wherein the sterilization temperature is about 95° C. andthe sterilization time is from about 1.5 hours to about 6.5 hours. 12.The sterile antiseptic solution of claim 1, wherein the sterilizationtemperature is about 110° C. and the sterilization time is from about 6minutes to about 90 minutes.
 13. The sterile antiseptic solution ofclaim 1, wherein the sterilization temperature is about 120° C. and thesterilization time is from about 2 minutes to about 35 minutes.
 14. Thesterile antiseptic solution of claim 1, wherein the container comprisesglass or plastic.
 15. The sterile antiseptic solution of claim 1,wherein the method further comprises the steps of: providing a cascadingwater sterilizer that produces a constant, cascading waterfall, whereinheating the antiseptic solution to the selected sterilizationtemperature and maintaining the selected sterilization temperaturecomprises contacting the container with the cascading waterfall for theduration of the sterilization time.
 16. The sterile antiseptic solutionof claim 1, wherein the method further comprises a step of cooling thesterile antiseptic solution after terminating the heating.
 17. Thesterile antiseptic solution of claim 1, wherein prior to heating theantiseptic solution to the selected sterilization temperature, thecontainer and the antiseptic solution have a temperature from about 20°C. to about 35° C.
 18. The sterile antiseptic solution of claim 1,wherein the container is sealed prior to heating the antiseptic solutionto the selected sterilization temperature.
 19. The sterile antisepticsolution of claim 7, further comprising water.
 20. The sterileantiseptic solution of claim 1, comprising from about 50% to about 80%v/v of the alcohol solvent, from about 0.1% to about 2.5% w/v of theantimicrobial molecule, and from about 10% to about 60% v/v water,wherein the alcoholic solvent and the water do not exceed 100% of atotal volume of the solution, and wherein the alcohol solvent isisopropanol and the antimicrobial molecule is chlorhexidine gluconate.21. The sterile antiseptic solution of claim 1, comprising about 0.1% ofthe antimicrobial molecule.
 22. The sterile antiseptic solution of claim1, wherein the antiseptic solution has an initial purity of at least98%.
 23. The sterile antiseptic solution of claim 1, wherein theselected sterilization temperature and the selected sterilization timeare chosen such that after terminating the heating, the sterileantiseptic solution has a post-sterilization purity of at least about94% and the percentage point change in purity from the initial purity tothe post-sterilization purity is at most about 4%.
 24. The sterileantiseptic solution of claim 1, wherein the selected sterilizationtemperature and the selected sterilization time are chosen such thatafter terminating the heating, the sterile antiseptic solution has apost-sterilization purity of at least about 96% and the percentage pointchange in purity from the initial purity to the post-sterilizationpurity is at most about 3%.
 25. The sterile antiseptic solution of claim1, wherein the selected sterilization temperature and the selectedsterilization time are chosen such that after terminating the heating,the sterile antiseptic solution has a post-sterilization purity of atleast about 98% and the percentage point change in purity from theinitial purity to the post-sterilization purity is at most about 2%. 26.The sterile antiseptic solution of claim 1, wherein the sterileantiseptic solution maintains 7-day sterility in accordance with U.S.Pharmacopeia Convention (USP) 36, Chapter 55, BiologicalIndicators—Resistance Performance Tests.